Mechanisms Of Drug-induced Toxicities

我们继续研究药物性肝病(DILD)的分子基础，这是一种罕见但往往危及生命的毒性。它是急性肝功能衰竭的主要原因，也是药物退出临床使用的主要原因。我们假设这种疾病的特殊性质部分是由于缺乏肝脏保护因子。今年，人们从以下几个方面对这一想法进行了探索： 1.去年我们报道了内源性IL-13对对乙酰氨基酚诱导的肝病(AILD)的保护作用，AILD是药物性肝功能衰竭的主要原因之一。今年，随着IL-13基因敲除小鼠和多重蛋白质芯片的使用，我们发现IL-13缺乏会导致几种促炎细胞因子和趋化因子的过度表达，这可能在确定小鼠甚至可能是人类对扑热息痛和其他药物引起的肝病的易感性方面发挥作用。 2.去年我们报道了解毒剂N-乙酰-L-半胱氨酸可能通过上调热休克蛋白、急性期蛋白和细胞周期调节基因等几种潜在的保肝因子来保护小鼠和可能的人类免受AILD的影响。今年，我们发现NAC在肝脏中诱导的急性时相蛋白之一是Lipocalin 2。我们目前正在确定这种急性时相蛋白的缺乏是由于给野生型小鼠注射中和抗体或使用Lipocalin 2敲除产生的，是否使小鼠更容易患AILD。 3.去年，我们利用同位素亲和标记(ICAT)质谱仪比较了AILD耐药株(SJL)和易感株(C57BL/6)小鼠肝脏蛋白质组，探讨了DILD保肝因子的多样性。结果显示，SJL小鼠体内有几种蛋白质水平较高，这些蛋白质可能会保护肝脏免受损伤。今年，我们通过比较这两个品系的小鼠和SJLxB6-F1杂交小鼠的肝脏mRNA表达谱来探索调节因子的多源性，SJLxB6-F1杂交小鼠被发现对AILD具有中等易感性。APAP治疗后24小时的整体肝脏基因表达谱显示，与敏感的B6小鼠相比，耐药的SJL小鼠中几个细胞保护基因的mRNA表达独特地升高，而SJLxB6-F1小鼠的这些基因的mRNA表达水平处于中等水平。其中一个基因编码热休克蛋白(HSP)70，其在三个品系小鼠中的相对蛋白表达水平与它们的mRNA水平平行，表明该蛋白对AILD具有保护作用。然而，与野生型小鼠相比，HSP70基因敲除小鼠对AILD的易感性没有差异。与SJL小鼠相比，B6小鼠中也有一些促炎基因，如骨桥蛋白，其mRNA表达水平高于SJL小鼠，而在SJLxB6-F1小鼠中则处于中等水平，这表明它们可能在APAP治疗后加剧肝损伤中发挥作用。为了支持这一观点，骨桥蛋白基因敲除小鼠被发现比野生型小鼠对AILD更具抵抗力。此外，蛋白质组和基因组研究的结果进行了比较，发现它们是相互补充的，而不是简单的重叠。 4.目前的证据表明，DILD通常是由肝脏药物-蛋白质加合物引起的过敏反应(药物性变态反应性肝炎，DIAH)引起的。Diah的发生率很低，并且无法在动物身上复制，这表明耐受机制可能会阻止Diah在大多数人和动物中发生。在这方面，去年我们发现肝毒性剂量的APAP与小鼠胸腺、脾和肝淋巴结中显著的淋巴细胞溶解有关。今年我们发现，在这个过程中，CD4和CD8单阳性T细胞以及CD4CD8双阳性T细胞和B细胞减少，从而导致适应性免疫系统的抑制。此外，淋巴细胞溶解的程度与血清皮质酮水平相关，表明这种应激激素在淋巴细胞溶解的机制中发挥作用。类似的过程也可能发生在人类身上，这有助于解释为什么Diah的发病率相对较低。

We have continued our studies of the molecular basis of drug-induced liver disease (DILD), which is a rare but often life-threatening toxicity. It is the major cause of acute liver failure and a principal reason drugs are withdrawn from clinical use. We have hypothesized that the idiosyncratic nature of this disease is due in part to a deficiency in hepatoprotective factors. This idea has been explored in the following ways this year: 1. Last year we reported that endogenous IL-13 can protect mice from acetaminophen-induced liver disease (AILD), which is a leading cause of drug-induced liver failure. This year with the use of IL-13 knockout mice and multiplex protein chips we have discovered that IL-13 deficiency leads to the over-expression of several proinflammatory cytokines and chemokines, which may have a role in determining the susceptibility of mice and possibly humans to liver disease caused by acetaminophen and other drugs. 2. Last year we reported that one of the ways the antidote N-acetyl-L-cysteine (NAC) may protect mice and possibly humans from AILD is by up-regulating several potential hepatoprotectic factors including heat shock proteins, acute phase proteins, and cell cycle regulation genes. This year we have found that one of the acute phase proteins induced by NAC in the liver is lipocalin 2. We are currently determining whether deficiency in this acute phase protein, produced by administering neutralizing antibodies to wild type mice or by the use of lipocalin 2 knockouts, makes mice more susceptible to AILD. 3. Last year we investigated the multiplicity of hepatoprotectant factors in DILD by comparing the liver proteomes of resistant (SJL) and susceptible (C57Bl/6) strains of mice to AILD with the use of isotope-coded affinity tag (ICAT) mass spectrometry. The results revealed that SJL mice had higher levels of several proteins that could potentially protect the liver from injury. This year we have explored the polygenicity of regulatory factors by comparing hepatic mRNA expression profiles of these two strains of mice with that of SJLxB6-F1 hybrid mice, which were found to be of intermediate susceptibility to AILD. Global hepatic gene expression profiling over a 24 h period following APAP treatment revealed a unique elevated pattern in the mRNA expression of several cytoprotective genes in resistant SJL mice compared to susceptible B6 mice, while SJLxB6-F1 mice had intermediate mRNA expression levels of these genes. One of these genes encoded for heat shock protein (HSP) 70 whose relative protein expression among the three strains of mice was found to parallel that of their mRNA levels, suggesting that this protein had a protective role against AILD. However, there was no difference in the susceptibility of HSP70 knockout mice to AILD compared to wild-type mice. There were also a number of pro-inflammatory genes, such as osteopontin, with elevated mRNA expression levels in the B6 mice compared to the SJL mice, and with intermediate levels in the SJLxB6-F1 mice, suggesting that they may play a role in exacerbating liver injury after APAP treatment. In support of this idea, osteopontin knockout mice were found to be more resistant to AILD than wild-type mice. Additionally, the results from both the proteomic and genomic studies were compared and found to be complementary to each other and not simply overlapping. 4. Current evidence indicates that DILD is often caused by an allergic response (drug-induced allergic hepatitis, DIAH) induced by hepatic drug-protein adducts. The low incidence of DIAH and inability to reproduce it in animals suggests that tolerogenic mechanisms may prevent DIAH from occurring in most people and animals. In this regard, last year we discovered that hepatotoxic doses of APAP were associated with marked lymphocytolysis in the thymus, spleen, and hepatic lymph nodes of mice. This year we have found that CD4 and CD8 single positive T cells as well CD4CD8 double positive T cells and B cells are diminished by this process, resulting in the inhibition of the adaptive immune system. Moreover, the degree of lymphocytolysis correlated with serum corticosterone levels, suggesting a role of this stress hormone in the mechanism of lymphocytolysis. Similar processes may occur in humans and help explain the relatively low incidence of DIAH.